Viscosity-reducing excipient compounds for protein formulations

ABSTRACT

The invention encompasses formulations and methods for the production thereof that permit the delivery of concentrated protein solutions. The inventive methods can yield a lower viscosity liquid formulation or a higher concentration of therapeutic or nontherapeutic proteins in the liquid formulation, as compared to traditional protein solutions.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 14/744,847filed on Jun. 19, 2015, which claims the benefit of U.S. ProvisionalApplication Ser. No. 62/014,784 filed Jun. 20, 2014, U.S. ProvisionalApplication No. 62/083,623, filed Nov. 24, 2014, and U.S. ProvisionalApplication Ser. No. 62/136,763 filed Mar. 23, 2015. The entire contentsof the each of the above applications are incorporated by referenceherein.

FIELD OF APPLICATION

This application relates generally to formulations for deliveringbiopolymers.

BACKGROUND

Biopolymers may be used for therapeutic or non-therapeutic purposes.Biopolymer-based therapeutics, such as antibody or enzyme formulations,are widely used in treating disease. Non-therapeutic biopolymers, suchas enzymes, peptides, and structural proteins, have utility innon-therapeutic applications such as household, nutrition, commercial,and industrial uses.

Biopolymers used in therapeutic applications must be formulated topermit their introduction into the body for treatment of disease. Forexample, it is advantageous to deliver antibody and protein/peptidebiopolymer formulations by subcutaneous (SC) or intramuscular (IM)routes under certain circumstances, instead of administering theseformulations by intravenous (IV) injections. In order to achieve betterpatient compliance and comfort with SC or IM injection though, theliquid volume in the syringe is typically limited to 2 to 3 ccs and theviscosity of the formulation is typically lower than about 20 centipoise(cP) so that the formulation can be delivered using conventional medicaldevices and small-bore needles. These delivery parameters do not alwaysfit well with the dosage requirements for the formulations beingdelivered.

Antibodies, for example, may need to be delivered at high dose levels toexert their intended therapeutic effect. Using a restricted liquidvolume to deliver a high dose level of an antibody formulation canrequire a high concentration of the antibody in the delivery vehicle,sometimes exceeding a level of 150 mg/mL. At this dosage level, theviscosity-versus-concentration plots of protein solutions lie beyondtheir linear-nonlinear transition, such that the viscosity of theformulation rises dramatically with increasing concentration. Increasedviscosity, however, is not compatible with standard SC or IM deliverysystems. The solutions of biopolymer-based therapeutics are also proneto stability problems, such as precipitation, hazing, opalescence,denaturing, and gel formation, reversible or irreversible aggregation.The stability problems limit the shelf life of the solutions or requirespecial handling.

One approach to producing protein formulations for injection is totransform the therapeutic protein solution into a powder that can bereconstituted to form a suspension suitable for SC or IM delivery.Lyophilization is a standard technique to produce protein powders.Freeze-drying, spray drying and even precipitation followed bysuper-critical-fluid extraction have been used to generate proteinpowders for subsequent reconstitution. Powdered suspensions are low inviscosity before re-dissolution (compared to solutions at the sameoverall dose) and thus may be suitable for SC or IM injection, providedthe particles are sufficiently small to fit through the needle. However,protein crystals that are present in the powder have the inherent riskof triggering immune response. The uncertain protein stability/activityfollowing re-dissolution poses further concerns. There remains a need inthe art for techniques to produce low viscosity protein formulations fortherapeutic purposes while avoiding the limitations introduced byprotein powder suspensions.

In addition to the therapeutic applications of proteins described above,biopolymers such as enzymes, peptides, and structural proteins can beused in non-therapeutic applications. These non-therapeutic biopolymerscan be produced from a number of different pathways, for example,derived from plant sources, animal sources, or produced from cellcultures.

The non-therapeutic proteins can be produced, transported, stored, andhandled as a granular or powdered material or as a solution ordispersion, usually in water. The biopolymers for non-therapeuticapplications can be globular or fibrous proteins, and certain forms ofthese materials can have limited solubility in water or exhibit highviscosity upon dissolution. These solution properties can presentchallenges to the formulation, handling, storage, pumping, andperformance of the non-therapeutic materials, so there is a need formethods to reduce viscosity and improve solubility and stability ofnon-therapeutic protein solutions.

Proteins are complex biopolymers, each with a uniquely folded 3-Dstructure and surface energy map (hydrophobic/hydrophilic regions andcharges). In concentrated protein solutions, these macromolecules maystrongly interact and even inter-lock in complicated ways, depending ontheir exact shape and surface energy distribution. “Hot-spots” forstrong specific interactions lead to protein clustering, increasingsolution viscosity. To address these concerns, a number of excipientcompounds are used in biotherapeutic formulations, aiming to reducesolution viscosity by impeding localized interactions and clustering.These efforts are individually tailored, often empirically, sometimesguided by in silico simulations. Combinations of excipient compounds maybe provided, but optimizing such combinations again must progressempirically and on a case-by case basis.

There remains a need in the art for a truly universal approach toreducing viscosity in protein formulations at a given concentrationunder nonlinear conditions. There is an additional need in the art toachieve this viscosity reduction while preserving the activity of theprotein. It would be further desirable to adapt the viscosity-reductionsystem to use with formulations having tunable and sustained releaseprofiles, and to use with formulations adapted for depot injection.

SUMMARY OF THE INVENTION

Disclosed herein, in embodiments, are liquid formulations comprising aprotein and an excipient compound selected from the group consisting ofhindered amines, anionic aromatics, functionalized amino acids,oligopeptides, short-chain organic acids, and low molecular weightaliphatic polyacids, wherein the excipient compound is added in aviscosity-reducing amount. In embodiments, the protein is a PEGylatedprotein and the excipient is a low molecular weight aliphatic polyacid.In embodiments, the formulation is a pharmaceutical composition, and thetherapeutic formulation comprises a therapeutic protein, wherein theexcipient compound is a pharmaceutically acceptable excipient compound.In embodiments, the formulation is a non-therapeutic formulation, andthe non-therapeutic formulation comprises a non-therapeutic protein. Inembodiments, the viscosity-reducing amount reduces viscosity of theformulation to a viscosity less than the viscosity of a controlformulation. In embodiments, the viscosity of the formulation is atleast about 10% less than the viscosity of the control formulation, oris at least about 30% less than the viscosity of the controlformulation, or is at least about 50% less than the viscosity of thecontrol formulation, or is at least about 70% less than the viscosity ofthe control formulation, or is at least about 90% less than theviscosity of the control formulation. In embodiments, the viscosity isless than about 100 cP, or is less than about 50 cP, or is less thanabout 20 cP, or is less than about 10 cP. In embodiments, the excipientcompound has a molecular weight of <5000 Da, or <1500 Da, or <500 Da. Inembodiments, the formulation contains at least about 25 mg/mL of theprotein, or at least about 100 mg/mL of the protein, or at least about200 mg/mL of the protein, or at least about 300 mg/mL of the protein. Inembodiments, the formulation comprises between about 5 mg/mL to about300 mg/mL of the excipient compound, or comprises between about 10 mg/mLto about 200 mg/mL of the excipient compound, or comprises between about20 mg/mL to about 100 mg/mL, or comprises between about 25 mg/mL toabout 75 mg/mL of the excipient compound. In embodiments, theformulation has an improved stability when compared to the controlformulation. In embodiments, the excipient compound is a hindered amine.In embodiments, the hindered amine is selected from the group consistingof caffeine, theophylline, tyramine, procaine, lidocaine, imidazole,aspartame, saccharin, and acesulfame potassium. In embodiments, thehindered amine is caffeine. In embodiments, the hindered amine is alocal injectable anesthetic compound. The hindered amine can possess anindependent pharmacological property, and the hindered amine can bepresent in the formulation in an amount that has an independentpharmacological effect. In embodiments the hindered amine can be presentin the formulation in an amount that is less than a therapeuticallyeffective amount. The independent pharmacological activity can be alocal anesthetic activity. In embodiments, the hindered amine possessingpossessing the independent pharmacological activity is combined with asecond excipient compound that further decreases the viscosity of theformulation. The second excipient compound can be selected from thegroup consisting of caffeine, theophylline, tyramine, procaine,lidocaine, imidazole, aspartame, saccharin, and acesulfame potassium. Inembodiments, the formulation can comprise an additional agent selectedfrom the group consisting of preservatives, surfactants, sugars,polysaccharides, arginine, proline, hyaluronidase, stabilizers, andbuffers.

Further disclosed herein are methods of treating a disease or disorderin a mammal, comprising administering to said mammal a liquidtherapeutic formulation, wherein the therapeutic formulation comprises atherapeutically effective amount of a therapeutic protein, and whereinthe formulation further comprises an pharmaceutically acceptableexcipient compound selected from the group consisting of hinderedamines, anionic aromatics, functionalized amino acids, oligopeptides,short-chain organic acids, and low molecular weight aliphatic polyacids;and wherein the therapeutic formulation is effective for the treatmentof the disease or disorder. In embodiments, the therapeutic protein is aPEGylated protein, and the excipient compound is a low molecular weightaliphatic polyacid. In embodiments, the excipient is a hindered amine.In embodiments, the hindered amine is a local anesthetic compound. Inembodiments, the formulation is administered by subcutaneous injection,or an intramuscular injection, or an intravenous injection. Inembodiments, the excipient compound is present in the therapeuticformulation in a viscosity-reducing amount, and the viscosity-reducingamount reduces viscosity of the therapeutic formulation to a viscosityless than the viscosity of a control formulation. In embodiments, thetherapeutic formulation has an improved stability when compared to thecontrol formulation. In embodiments, the excipient compound isessentially pure.

Further disclosed herein are methods of reducing pain at an injectionsite of a therapeutic protein in a mammal in need thereof, comprising:administering a liquid therapeutic formulation by injection, wherein thetherapeutic formulation comprises a therapeutically effective amount ofthe therapeutic protein, wherein the formulation further comprises anpharmaceutically acceptable excipient compound selected from the groupconsisting of local injectable anesthetic compounds, wherein thepharmaceutically acceptable excipient compound is added to theformulation in a viscosity-reducing amount; and wherein the mammalexperiences less pain with administration of the therapeutic formulationcomprising the excipient compound than that with administration of acontrol therapeutic formulation, wherein the control therapeuticformulation does not contain the excipient compound and is otherwiseidentical to the therapeutic formulation.

Disclosed herein, in embodiments, are methods of improving stability ofa liquid protein formulation, comprising: preparing a liquid proteinformulation comprising a therapeutic protein and an excipient compoundselected from the group selected from the group consisting of hinderedamines, anionic aromatics, functionalized amino acids, oligopeptides,and short-chain organic acids, and low molecular weight aliphaticpolyacids, wherein the liquid protein formulation demonstrates improvedstability compared to a control liquid protein formulation, wherein thecontrol liquid protein formulation does not contain the excipientcompound and is otherwise identical to the liquid protein formulation.The stability of the liquid formulation can be a cold storage conditionsstability, a room temperature stability or an elevated temperaturestability.

Also disclosed herein, in embodiments, are liquid formulationscomprising a protein and an excipient compound selected from the groupconsisting of hindered amines, anionic aromatics, functionalized aminoacids, oligopeptides, short-chain organic acids, and low molecularweight aliphatic polyacids, wherein the presence of the excipientcompound in the formulation results in improved protein-proteininteraction characteristics as measured by the protein diffusioninteraction parameter kD, or the second virial coefficient B22. Inembodiments, the formulation is a therapeutic formulation, and comprisesa therapeutic protein. In embodiments, the formulation is anon-therapeutic formulation, and comprises a non-therapeutic protein.

Further disclosed herein, in embodiments, are methods of improving aprotein-related process comprising providing the liquid formulationdescribed above, and employing it in a processing method. Inembodiments, the processing method includes filtration, pumping, mixing,centrifugation, membrane separation, lyophilization, or chromatography.

DETAILED DESCRIPTION

Disclosed herein are formulations and methods for their production thatpermit the delivery of concentrated protein solutions. In embodiments,the approaches disclosed herein can yield a lower viscosity liquidformulation or a higher concentration of therapeutic or nontherapeuticproteins in the liquid formulation, as compared to traditional proteinsolutions. In embodiments, the approaches disclosed herein can yield aliquid formulation having improved stability when compared to atraditional protein solution. A stable formulation is one in which theprotein contained therein substantially retains its physical andchemical stability and its therapeutic or nontherapeutic efficacy uponstorage under storage conditions, whether cold storage conditions, roomtemperature conditions, or elevated temperature storage conditions.Advantageously, a stable formulation can also offer protection againstaggregation or precipitation of the proteins dissolved therein. Forexample, the cold storage conditions can entail storage in arefrigerator or freezer. In some examples, cold storage conditions canentail conventional refrigerator or freezer storage at a temperature of10° C. or less. In additional examples, the cold storage conditionsentail storage at a temperature from about 2° to about 10° C. In otherexamples, the cold storage conditions entail storage at a temperature ofabout 4° C. In additional examples, the cold storage conditions entailstorage at freezing temperature such as about 0° C. or lower. In anotherexample, cold storage conditions entail storage at a temperature ofabout −30° C. to about 0° C. The room temperature storage conditions canentail storage at ambient temperatures, for example, from about 10° C.to about 30° C. Elevated temperature stability, for example, attemperatures from about 30° C. to about 50° C., can be used as part ofan accelerated aging study to predict the long term storage at typicalambient (10-30° C.) conditions.

It is well known to those skilled in the art of polymer science andengineering that proteins in solution tend to form entanglements, whichcan limit the translational mobility of the entangled chains andinterfere with the protein's therapeutic or nontherapeutic efficacy. Inembodiments, excipient compounds as disclosed herein can suppressprotein clustering due to specific interactions between the excipientcompound and the target protein in solution. Excipient compounds asdisclosed herein can be natural or synthetic, and desirably aresubstances that the FDA generally recognizes as safe (“GRAS”).

1. Definitions

For the purpose of this disclosure, the term “protein” refers to asequence of amino acids having a chain length long enough to produce adiscrete tertiary structure, typically having a molecular weight between1-3000 kD. In some embodiments, the molecular weight of the protein isbetween about 50-200 kD; in other embodiments, the molecular weight ofthe protein is between about 20-1000 kD or between about 20-2000 kD. Incontrast to the term “protein,” the term “peptide” refers to a sequenceof amino acids that does not have a discrete tertiary structure. A widevariety of biopolymers are included within the scope of the term“protein.” For example, the term “protein” can refer to therapeutic ornon-therapeutic proteins, including antibodies, aptamers, fusionproteins, PEGylated proteins, synthetic polypeptides, protein fragments,lipoproteins, enzymes, structural peptides, and the like.

As non-limiting examples, therapeutic proteins can include mammalianproteins such as hormones and prohormones (e.g., insulin and proinsulin,glucagon, calcitonin, thyroid hormones (T3 or T4 or thyroid-stimulatinghormone), parathyroid hormone, follicle-stimulating hormone, luteinizinghormone, growth hormone, growth hormone releasing factor, and the like);clotting and anti-clotting factors (e.g., tissue factor, vonWillebrand's factor, Factor VIIIC, Factor IX, protein C, plasminogenactivators (urokinase, tissue-type plasminogen activators), thrombin);cytokines, chemokines, and inflammatory mediators; interferons;colony-stimulating factors; interleukins (e.g., IL-1 through IL-10);growth factors (e.g., vascular endothelial growth factors, fibroblastgrowth factor, platelet-derived growth factor, transforming growthfactor, neurotrophic growth factors, insulin-like growth factor, and thelike); albumins; collagens and elastins; hematopoietic factors (e.g.,erythropoietin, thrombopoietin, and the like); osteoinductive factors(e.g., bone morphogenetic protein); receptors (e.g., integrins,cadherins, and the like); surface membrane proteins; transport proteins;regulatory proteins; antigenic proteins (e.g., a viral component thatacts as an antigen); and antibodies. The term “antibody” is used hereinin its broadest sense, to include as non-limiting examples monoclonalantibodies (including, for example, full-length antibodies with animmunoglobulin Fc region), single-chain molecules, bi-specific andmulti-specific antibodies, diabodies, antibody compositions havingpolyepitopic specificity, and fragments of antibodies (including, forexample, Fab, Fv, and F(ab′)2). Antibodies can also be termed“immunoglobulins.” An antibody is understood to be directed against aspecific protein or non-protein “antigen,” which is a biologicallyimportant material; the administration of a therapeutically effectiveamount of an antibody to a patient can complex with the antigen, therebyaltering its biological properties so that the patient experiences atherapeutic effect.

In embodiments, the proteins are PEGylated, meaning that they comprisepoly(ethylene glycol) (“PEG”) and/or poly(propylene glycol) (“PPG”)units. PEGylated proteins, or PEG-protein conjugates, have found utilityin therapeutic applications due to their beneficial properties such assolubility, pharmacokinetics, pharmacodynamics, immunogenicity, renalclearance, and stability. Non-limiting examples of PEGylated proteinsare PEGylated interferons (PEG-IFN), PEGylated anti-VEGF, PEG proteinconjugate drugs, Adagen, Pegaspargase, Pegfilgrastim, Pegloticase,Pegvisomant, PEGylated epoetin-β, and Certolizumab pegol.

PEGylated proteins can be synthesized by a variety of methods such as areaction of protein with a PEG reagent having one or more reactivefunctional groups. The reactive functional groups on the PEG reagent canform a linkage with the protein at targeted protein sites such aslysine, histidine, cysteine, and the N-terminus. Typical PEGylationreagents have reactive functional groups such as aldehyde, maleimide, orsuccinimide groups that have specific reactivity with targeted aminoacid residues on proteins. The PEGylation reagents can have a PEG chainlength from about 1 to about 1000 PEG and/or PPG repeating units. Othermethods of PEGylation include glyco-PEGylation, where the protein isfirst glycosylated and then the glycosylated residues are PEGylated in asecond step. Certain PEGylation processes are assisted by enzymes likesialyltransferase and transglutaminase.

While the PEGylated proteins can offer therapeutic advantages overnative, non-PEGylated proteins, these materials can have physical orchemical properties that make them difficult to purify, dissolve,filter, concentrate, and administer. The PEGylation of a protein canlead to a higher solution viscosity compared to the native protein, andthis generally requires the formulation of PEGylated protein solutionsat lower concentrations.

It is desirable to formulate protein therapeutics in stable, lowviscosity solutions so they can be administered to patients in a minimalinjection volume. For example, the subcutaneous (SC) or intramuscular(IM) injection of drugs generally requires a small injection volume,preferably less than 2 mL. The SC and IM injection routes are wellsuited to self-administered care, and this is a less costly and moreaccessible form of treatment compared with intravenous (IV) injectionwhich is only conducted under direct medical supervision. Formulationsfor SC or IM injection require a low solution viscosity, generally below30 cP, and preferably below 20 cP, to allow easy flow of the therapeuticsolution through a narrow gauge needle. This combination of smallinjection volume and low viscosity requirements present a challenge tothe use of PEGylated protein therapeutics in SC or IM injection routes.

Those proteins having therapeutic effects may be termed “therapeuticproteins”; formulations containing therapeutic proteins intherapeutically effective amounts may be termed “therapeuticformulations.” The therapeutic protein contained in a therapeuticformulation may also be termed its “protein active ingredient.”Typically, a therapeutic formulation comprises a therapeuticallyeffective amount of a protein active ingredient and an excipient, withor without other optional components. As used herein, the term“therapeutic” includes both treatments of existing disorders andpreventions of disorders.

A “treatment” includes any measure intended to cure, heal, alleviate,improve, remedy, or otherwise beneficially affect the disorder,including preventing or delaying the onset of symptoms and/oralleviating or ameliorating symptoms of the disorder. Those patients inneed of a treatment include both those who already have a specificdisorder, and those for whom the prevention of a disorder is desirable.A disorder is any condition that alters the homeostatic wellbeing of amammal, including acute or chronic diseases, or pathological conditionsthat predispose the mammal to an acute or chronic disease. Non-limitingexamples of disorders include cancers, metabolic disorders (e.g.,diabetes), allergic disorders (e.g., asthma), dermatological disorders,cardiovascular disorders, respiratory disorders, hematologicaldisorders, musculoskeletal disorders, inflammatory or rheumatologicaldisorders, autoimmune disorders, gastrointestinal disorders, urologicaldisorders, sexual and reproductive disorders, neurological disorders,and the like. The term “mammal” for the purposes of treatment can referto any animal classified as a mammal, including humans, domesticanimals, pet animals, farm animals, sporting animals, working animals,and the like. A “treatment” can therefore include both veterinary andhuman treatments. For convenience, the mammal undergoing such“treatment” can be referred to as a “patient.” In certain embodiments,the patient can be of any age, including fetal animals in utero.

In embodiments, a treatment involves providing a therapeuticallyeffective amount of a therapeutic formulation to a mammal in needthereof. A “therapeutically effective amount” is at least the minimumconcentration of the therapeutic protein administered to the mammal inneed thereof, to effect a treatment of an existing disorder or aprevention of an anticipated disorder (either such treatment or suchprevention being a “therapeutic intervention”). Therapeuticallyeffective amounts of various therapeutic proteins that may be includedas active ingredients in the therapeutic formulation may be familiar inthe art; or, for therapeutic proteins discovered or applied totherapeutic interventions hereinafter, the therapeutically effectiveamount can be determined by standard techniques carried out by thosehaving ordinary skill in the art, using no more than routineexperimentation.

Those proteins used for non-therapeutic purposes (i.e., purposes notinvolving treatments), such as household, nutrition, commercial, andindustrial applications, may be termed “non-therapeutic proteins.”Formulations containing non-therapeutic proteins may be termed“non-therapeutic formulations”. The non-therapeutic proteins can bederived from plant sources, animal sources, or produced from cellcultures; they also can be enzymes or structural proteins. Thenon-therapeutic proteins can be used in in household, nutrition,commercial, and industrial applications such as catalysts, human andanimal nutrition, processing aids, cleaners, and waste treatment.

An important category of non-therapeutic biopolymers is enzymes. Enzymeshave a number of non-therapeutic applications, for example, ascatalysts, human and animal nutritional ingredients, processing aids,cleaners, and waste treatment agents. Enzyme catalysts are used toaccelerate a variety of chemical reactions. Examples of enzyme catalystsfor non-therapeutic uses include catalases, oxidoreductases,transferases, hydrolases, lyases, isomerases, and ligases. Human andanimal nutritional uses of enzymes include nutraceuticals, nutritivesources of protein, chelation or controlled delivery of micronutrients,digestion aids, and supplements; these can be derived from amylase,protease, trypsin, lactase, and the like. Enzymatic processing aids areused to improve the production of food and beverage products inoperations like baking, brewing, fermenting, juice processing, andwinemaking. Examples of these food and beverage processing aids includeamylases, cellulases, pectinases, glucanases, lipases, and lactases.Enzymes can also be used in the production of biofuels. Ethanol forbiofuels, for example, can be aided by the enzymatic degradation ofbiomass feedstocks such as cellulosic and lignocellulosic materials. Thetreatment of such feedstock materials with cellulases and ligninasestransforms the biomass into a substrate that can be fermented intofuels. In other commercial applications, enzymes are used as detergents,cleaners, and stain lifting aids for laundry, dish washing, surfacecleaning, and equipment cleaning applications. Typical enzymes for thispurpose include proteases, cellulases, amylases, and lipases. Inaddition, non-therapeutic enzymes are used in a variety of commercialand industrial processes such as textile softening with cellulases,leather processing, waste treatment, contaminated sediment treatment,water treatment, pulp bleaching, and pulp softening and debonding.Typical enzymes for these purposes are amylases, xylanases, cellulases,and ligninases.

Other examples of non-therapeutic biopolymers include fibrous orstructural proteins such as keratins, collagen, gelatin, elastin,fibroin, actin, tubulin, or the hydrolyzed, degraded, or derivatizedforms thereof. These materials are used in the preparation andformulation of food ingredients such as gelatin, ice cream, yogurt, andconfections; they area also added to foods as thickeners, rheologymodifiers, mouthfeel improvers, and as a source of nutritional protein.In the cosmetics and personal care industry, collagen, elastin, keratin,and hydrolyzed keratin are widely used as ingredients in skin care andhair care formulations. Still other examples of non-therapeuticbiopolymers are whey proteins such as beta-lactoglobulin,alpha-lactalbumin, and serum albumin. These whey proteins are producedin mass scale as a byproduct from dairy operations and have been usedfor a variety of non-therapeutic applications.

2. Therapeutic Formulations

In one aspect, the formulations and methods disclosed herein providestable liquid formulations of improved or reduced viscosity, comprisinga therapeutic protein in a therapeutically effective amount and anexcipient compound. In embodiments, the formulation can improve thestability while providing an acceptable concentration of activeingredients and an acceptable viscosity. In embodiments, the formulationprovides an improvement in stability when compared to a controlformulation; for the purposes of this disclosure, a control formulationis a formulation containing the protein active ingredient that isidentical on a dry weight basis in every way to the therapeuticformulation except that it lacks the excipient compound. In embodiments,improved stability of the protein containing formulation is in the formof lower percentage of soluble aggregates, particulates, subvisibleparticles, or gel formation, compared to a control formulation.

It is understood that the viscosity of a liquid protein formulation canbe affected by a variety of factors, including but not limited to: thenature of the protein itself (e.g., enzyme, antibody, receptor, fusionprotein, etc.); its size, three-dimensional structure, chemicalcomposition, and molecular weight; its concentration in the formulation;the components of the formulation besides the protein; the desired pHrange; the storage conditions for the formulation; and the method ofadministering the formulation to the patient. Therapeutic proteins mostsuitable for use with the excipient compounds described herein arepreferably essentially pure, i.e., free from contaminating proteins. Inembodiments, an “essentially pure” therapeutic protein is a proteincomposition comprising at least 90% by weight of the therapeuticprotein, or preferably at least 95% by weight, or more preferably, atleast 99% by weight, all based on the total weight of therapeuticproteins and contaminating proteins in the composition. For the purposesof clarity, a protein added as an excipient is not intended to beincluded in this definition. The therapeutic formulations describedherein are intended for use as pharmaceutical-grade formulations, i.e.,formulations intended for use in treating a mammal, in such a form thatthe desired therapeutic efficacy of the protein active ingredient can beachieved, and without containing components that are toxic to the mammalto whom the formulation is to be administered.

In embodiments, the therapeutic formulation contains at least 25 mg/mLof protein active ingredient. In other embodiments, the therapeuticformulation contains at least 100 mg/mL of protein active ingredient. Inother embodiments, the therapeutic formulation contains at least 200mg/mL of protein active ingredient. In yet other embodiments, thetherapeutic formulation solution contains at least 300 mg/mL of proteinactive ingredient. Generally, the excipient compounds disclosed hereinare added to the therapeutic formulation in an amount between about 5 toabout 300 mg/mL. In embodiments, the excipient compound can be added inan amount of about 10 to about 200 mg/mL. In embodiments, the excipientcompound can be added in an amount of about 20 to about 100 mg/mL. Inembodiments, the excipient can be added in an amount of about 25 toabout 75 mg/mL.

Excipient compounds of various molecular weights are selected forspecific advantageous properties when combined with the protein activeingredient in a formulation. Examples of therapeutic formulationscomprising excipient compounds are provided below. In embodiments, theexcipient compound has a molecular weight of <5000 Da. In embodiments,the excipient compound has a molecular weight of <1000 Da. Inembodiments, the excipient compound has a molecular weight of <500 Da.

In embodiments, the excipient compounds disclosed herein is added to thetherapeutic formulation in a viscosity-reducing amount. In embodiments,a viscosity-reducing amount is the amount of an excipient compound thatreduces the viscosity of the formulation at least 10% when compared to acontrol formulation; for the purposes of this disclosure, a controlformulation is a formulation containing the protein active ingredientthat is identical on a dry weight basis in every way to the therapeuticformulation except that it lacks the excipient compound. In embodiments,the viscosity-reducing amount is the amount of an excipient compoundthat reduces the viscosity of the formulation at least 30% when comparedto the control formulation. In embodiments, the viscosity-reducingamount is the amount of an excipient compound that reduces the viscosityof the formulation at least 50% when compared to the controlformulation. In embodiments, the viscosity-reducing amount is the amountof an excipient compound that reduces the viscosity of the formulationat least 70% when compared to the control formulation. In embodiments,the viscosity-reducing amount is the amount of an excipient compoundthat reduces the viscosity of the formulation at least 90% when comparedto the control formulation.

In embodiments, the viscosity-reducing amount yields a therapeuticformulation having a viscosity of less than 100 cP. In otherembodiments, the therapeutic formulation has a viscosity of less than 50cP. In other embodiments, the therapeutic formulation has a viscosity ofless than 20 cP. In yet other embodiments, the therapeutic formulationhas a viscosity of less than 10 cP. The term “viscosity” as used hereinrefers to a dynamic viscosity value when measured by the methodsdisclosed herein.

Therapeutic formulations in accordance with this disclosure have certainadvantageous properties. In embodiments, the therapeutic formulationsare resistant to shear degradation, phase separation, clouding out,precipitation, and denaturing. In embodiments, the therapeuticformulations are processed, purified, stored, syringed, dosed, filtered,and centrifuged more effectively, compared with a control formulation.In embodiments, the therapeutic formulations are administered to apatient at high concentration of therapeutic protein. In embodiments,the therapeutic formulations are administered to patients with lessdiscomfort than would be experienced with a similar formulation lackingthe therapeutic excipient. In embodiments, the therapeutic formulationsare administered as a depot injection. In embodiments, the therapeuticformulations extend the half-life of the therapeutic protein in thebody. These features of therapeutic formulations as disclosed hereinwould permit the administration of such formulations by intramuscular orsubcutaneous injection in a clinical situation, i.e., a situation wherepatient acceptance of an intramuscular injection would include the useof small-bore needles typical for IM/SC purposes and the use of atolerable (for example, 2-3 cc) injected volume, and where theseconditions result in the administration of an effective amount of theformulation in a single injection at a single injection site. Bycontrast, injection of a comparable dosage amount of the therapeuticprotein using conventional formulation techniques would be limited bythe higher viscosity of the conventional formulation, so that a SC/IMinjection of the conventional formulation would not be suitable for aclinical situation.

In embodiments, the therapeutic excipient has antioxidant propertiesthat stabilize the therapeutic protein against oxidative damage. Inembodiments, the therapeutic formulation is stored at ambienttemperatures, or for extended time at refrigerator conditions withoutappreciable loss of potency for the therapeutic protein. In embodiments,the therapeutic formulation is dried down for storage until it isneeded; then it is reconstituted with an appropriate solvent, e.g.,water. Advantageously, the formulations prepared as described herein canbe stable over a prolonged period of time, from months to years. Whenexceptionally long periods of storage are desired, the formulations canbe preserved in a freezer (and later reactivated) without fear ofprotein denaturation. In embodiments, formulations can be prepared forlong-term storage that do not require refrigeration.

Methods for preparing therapeutic formulations may be familiar toskilled artisans. The therapeutic formulations of the present inventioncan be prepared, for example, by adding the excipient compound to theformulation before or after the therapeutic protein is added to thesolution. The therapeutic formulation can, for example, be produced bycombining the therapeutic protein and the excipient at a first (lower)concentration and then processed by filtration or centrifugation toproduce a second (higher) concentration of the therapeutic protein.Therapeutic formulations can be made with one or more of the excipientcompounds with chaotropes, kosmotropes, hydrotropes, and salts.Therapeutic formulations can be made with one or more of the excipientcompounds using techniques such as encapsulation, dispersion, liposome,vesicle formation, and the like. Methods for preparing therapeuticformulations comprising the excipient compounds disclosed herein caninclude combinations of the excipient compounds. In embodiments,combinations of excipients can produce benefits in lower viscosity,improved stability, or reduced injection site pain. Other additives maybe introduced into the therapeutic formulations during theirmanufacture, including preservatives, surfactants, sugars, sucrose,trehalose, polysaccharides, arginine, proline, hyaluronidase,stabilizers, buffers, and the like. As used herein, a pharmaceuticallyacceptable excipient compound is one that is non-toxic and suitable foranimal and/or human administration.

3. Non-Therapeutic Formulations

In one aspect, the formulations and methods disclosed herein providestable liquid formulations of improved or reduced viscosity, comprisinga non-therapeutic protein in an effective amount and an excipientcompound. In embodiments, the formulation improves the stability whileproviding an acceptable concentration of active ingredients and anacceptable viscosity. In embodiments, the formulation provides animprovement in stability when compared to a control formulation; for thepurposes of this disclosure, a control formulation is a formulationcontaining the protein active ingredient that is identical on a dryweight basis in every way to the non-therapeutic formulation except thatit lacks the excipient compound.

It is understood that the viscosity of a liquid protein formulation canbe affected by a variety of factors, including but not limited to: thenature of the protein itself (e.g., enzyme, structural protein, degreeof hydrolysis, etc.); its size, three-dimensional structure, chemicalcomposition, and molecular weight; its concentration in the formulation;the components of the formulation besides the protein; the desired pHrange; and the storage conditions for the formulation.

In embodiments, the non-therapeutic formulation contains at least 25mg/mL of protein active ingredient. In other embodiments, thenon-therapeutic formulation contains at least 100 mg/mL of proteinactive ingredient. In other embodiments, the non-therapeutic formulationcontains at least 200 mg/mL of protein active ingredient. In yet otherembodiments, the non-therapeutic formulation solution contains at least300 mg/mL of protein active ingredient. Generally, the excipientcompounds disclosed herein are added to the non-therapeutic formulationin an amount between about 5 to about 300 mg/mL. In embodiments, theexcipient compound is added in an amount of about 10 to about 200 mg/mL.In embodiments, the excipient compound is added in an amount of about 20to about 100 mg/mL. In embodiments, the excipient is added in an amountof about 25 to about 75 mg/mL.

Excipient compounds of various molecular weights are selected forspecific advantageous properties when combined with the protein activeingredient in a formulation. Examples of non-therapeutic formulationscomprising excipient compounds are provided below. In embodiments, theexcipient compound has a molecular weight of <5000 Da. In embodiments,the excipient compound has a molecular weight of <1000 Da. Inembodiments, the excipient compound has a molecular weight of <500 Da.

In embodiments, the excipient compounds disclosed herein is added to thenon-therapeutic formulation in a viscosity-reducing amount. Inembodiments, a viscosity-reducing amount is the amount of an excipientcompound that reduces the viscosity of the formulation at least 10% whencompared to a control formulation; for the purposes of this disclosure,a control formulation is a formulation containing the protein activeingredient that is identical on a dry weight basis in every way to thetherapeutic formulation except that it lacks the excipient compound. Inembodiments, the viscosity-reducing amount is the amount of an excipientcompound that reduces the viscosity of the formulation at least 30% whencompared to the control formulation. In embodiments, theviscosity-reducing amount is the amount of an excipient compound thatreduces the viscosity of the formulation at least 50% when compared tothe control formulation. In embodiments, the viscosity-reducing amountis the amount of an excipient compound that reduces the viscosity of theformulation at least 70% when compared to the control formulation. Inembodiments, the viscosity-reducing amount is the amount of an excipientcompound that reduces the viscosity of the formulation at least 90% whencompared to the control formulation.

In embodiments, the viscosity-reducing amount yields a non-therapeuticformulation having a viscosity of less than 100 cP. In otherembodiments, the non-therapeutic formulation has a viscosity of lessthan 50 cP. In other embodiments, the non-therapeutic formulation has aviscosity of less than 20 cP. In yet other embodiments, thenon-therapeutic formulation has a viscosity of less than 10 cP. The term“viscosity” as used herein refers to a dynamic viscosity value.

Non-therapeutic formulations in accordance with this disclosure can havecertain advantageous properties. In embodiments, the non-therapeuticformulations are resistant to shear degradation, phase separation,clouding out, precipitation, and denaturing. In embodiments, thetherapeutic formulations can be processed, purified, stored, pumped,filtered, and centrifuged more effectively, compared with a controlformulation.

In embodiments, the non-therapeutic excipient has antioxidant propertiesthat stabilize the non-therapeutic protein against oxidative damage. Inembodiments, the non-therapeutic formulation is stored at ambienttemperatures, or for extended time at refrigerator conditions withoutappreciable loss of potency for the non-therapeutic protein. Inembodiments, the non-therapeutic formulation is dried down for storageuntil it is needed; then it can be reconstituted with an appropriatesolvent, e.g., water. Advantageously, the formulations prepared asdescribed herein is stable over a prolonged period of time, from monthsto years. When exceptionally long periods of storage are desired, theformulations are preserved in a freezer (and later reactivated) withoutfear of protein denaturation. In embodiments, formulations are preparedfor long-term storage that do not require refrigeration.

Methods for preparing non-therapeutic formulations comprising theexcipient compounds disclosed herein may be familiar to skilledartisans. For example, the excipient compound can be added to theformulation before or after the non-therapeutic protein is added to thesolution. The non-therapeutic formulation can be produced at a first(lower) concentration and then processed by filtration or centrifugationto produce a second (higher) concentration. Non-therapeutic formulationscan be made with one or more of the excipient compounds with chaotropes,kosmotropes, hydrotropes, and salts. Non-therapeutic formulations can bemade with one or more of the excipient compounds using techniques suchas encapsulation, dispersion, liposome, vesicle formation, and the like.Other additives can be introduced into the non-therapeutic formulationsduring their manufacture, including preservatives, surfactants,stabilizers, and the like.

4. Excipient Compounds

Several excipient compounds are described herein, each suitable for usewith one or more therapeutic or non-therapeutic proteins, and eachallowing the formulation to be composed so that it contains theprotein(s) at a high concentration. Some of the categories of excipientcompounds described below are: (1) hindered amines; (2) anionicaromatics; (3) functionalized amino acids; and (4) oligopeptides.Without being bound by theory, the excipient compounds described hereinare thought to associate with certain fragments, sequences, structures,or sections of a therapeutic protein that otherwise would be involved ininter-particle (i.e., protein-protein) interactions. The association ofthese excipient compounds with the therapeutic or non-therapeuticprotein can mask the inter-protein interactions such that the proteinscan be formulated in high concentration without causing excessivesolution viscosity. Excipient compounds advantageously can bewater-soluble, therefore suitable for use with aqueous vehicles. Inembodiments, the excipient compounds have a water solubility of >10mg/mL. In embodiments, the excipient compounds have a water solubilityof >100 mg/mL. In embodiments, the excipient compounds have a watersolubility of >500 mg/mL. Advantageously for therapeutic proteins, theexcipient compounds can be derived from materials that are biologicallyacceptable and are non-immunogenic, and are thus suitable forpharmaceutical use. In therapeutic embodiments, the excipient compoundscan be metabolized in the body to yield biologically compatible andnon-immunogenic byproducts.

a. Excipient Compound Category 1: Hindered Amines

High concentration solutions of therapeutic or non-therapeutic proteinscan be formulated with hindered amine small molecules as excipientcompounds. As used herein, the term “hindered amine” refers to a smallmolecule containing at least one bulky or sterically hindered group,consistent with the examples below. Hindered amines can be used in thefree base form, in the protonated form, or a combination of the two. Inprotonated forms, the hindered amines can be associated with an anioniccounterion such as chloride, hydroxide, bromide, iodide, fluoride,acetate, formate, phosphate, sulfate, or carboxylate. Hindered aminecompounds useful as excipient compounds can contain secondary amine,tertiary amine, quaternary ammonium, pyridinium, pyrrolidone,pyrrolidine, piperidine, morpholine, or guanidinium groups, such thatthe excipient compound has a cationic charge in aqueous solution atneutral pH. The hindered amine compounds also contain at least one bulkyor sterically hindered group, such as cyclic aromatic, cycloaliphatic,cyclohexyl, or alkyl groups. In embodiments, the sterically hinderedgroup can itself be an amine group such as a dialkylamine,trialkylamine, guanidinium, pyridinium, or quaternary ammonium group.Without being bound by theory, the hindered amine compounds are thoughtto associate with aromatic sections of the proteins such asphenylalanine, tryptophan, and tyrosine, by a cation pi interaction. Inembodiments, the cationic group of the hindered amine can have anaffinity for the electron rich pi structure of the aromatic amino acidresidues in the protein, so that they can shield these sections of theprotein, thereby decreasing the tendency of such shielded proteins toassociate and agglomerate.

In embodiments, the hindered amine excipient compounds has a chemicalstructure comprising imidazole, imidazoline, or imidazolidine groups, orsalts thereof, such as imidazole, 1-methylimidazole, 4-methylimidazole,1-hexyl-3-methylimidazolium chloride, histamine, 4-methylhistamine,alpha-methylhistamine, betahistine, beta-alanine,2-methyl-2-imidazoline, 1-butyl-3-methylimidazolium chloride, uric acid,potassium urate, betazole, carnosine, aspartame, saccharin, acesulfamepotassium, xanthine, theophylline, theobromine, caffeine, and anserine.In embodiments, the hindered amine excipient compounds is selected fromthe group consisting of dimethylethanolamine, dimethylaminopropylamine,triethanolamine, dimethylbenzylamine, dimethylcyclohexylamine,diethylcyclohexylamine, dicyclohexylmethylamine, hexamethylenebiguanide, poly(hexamethylene biguanide), imidazole, dimethylglycine,agmatine, diazabicyclo[2.2.2]octane, tetramethylethylenediamine,N,N-dimethylethanolamine, ethanolamine phosphate, glucosamine, cholinechloride, phosphocholine, niacinamide, isonicotinamide, N,N-diethylnicotinamide, nicotinic acid sodium salt, tyramine, 3-aminopyridine,2,4,6-trimethylpyridine, 3-pyridine methanol, nicotinamide adenosinedinucleotide, biotin, morpholine, N-methylpyrrolidone, 2-pyrrolidinone,procaine, lidocaine, dicyandiamide-taurine adduct, 2-pyridylethylamine,dicyandiamide-benzyl amine adduct, dicyandiamide-alkylamine adduct,dicyandiamide-cycloalkylamine adduct, anddicyandiamide-aminomethanephosphonic acid adducts. In embodiments, ahindered amine compound consistent with this disclosure is formulated asa protonated ammonium salt. In embodiments, a hindered amine compoundconsistent with this disclosure is formulated as a salt with aninorganic anion or organic anion as the counterion. In embodiments, highconcentration solutions of therapeutic or non-therapeutic proteins areformulated with a combination of caffeine with a benzoic acid, ahydroxybenzoic acid, or a benzenesulfonic acid as excipient compounds.In embodiments, the hindered amine excipient compounds is metabolized inthe body to yield biologically compatible byproducts. In someembodiments, the hindered amine excipient compound is present in theformulation at a concentration of about 250 mg/ml or less. In additionalembodiments, the hindered amine excipient compound is present in theformulation at a concentration of about 10 mg/ml to about 200 mg/ml. Inyet additional aspects, the hindered amine excipient compound is presentin the formulation at a concentration of about 20 to about 120 mg/ml.

In embodiments, certain hindered amine excipient compounds can possessother pharmacological properties. As examples, xanthines are a categoryof hindered amines having independent pharmacological properties,including stimulant properties and bronchodilator properties whensystemically absorbed. Representative xanthines include caffeine,aminophylline, 3-isobutyl-1-methylxanthine, paraxanthine,pentoxifylline, theobromine, theophylline, and the like. Methylatedxanthines are understood to affect force of cardiac contraction, heartrate, and bronchodilation. In some embodiments, the xanthine excipientcompound is present in the formulation at a concentration of about 30mg/ml or less.

Another category of hindered amines having independent pharmacologicalproperties are the local injectable anesthetic compounds. Localinjectable anesthetic compounds are hindered amines that have athree-component molecular structure of (a) a lipophilic aromatic ring,(b) an intermediate ester or amide linkage, and (c) a secondary ortertiary amine. This category of hindered amines is understood tointerrupt neural conduction by inhibiting the influx of sodium ions,thereby inducing local anesthesia. The lipophilic aromatic ring for alocal anesthetic compound may be formed of carbon atoms (e.g., a benzenering) or it may comprise heteroatoms (e.g., a thiophene ring).Representative local injectable anesthetic compounds include, but arenot limited to, amylocaine, articaine, bupivicaine, butacaine,butanilicaine, chlorprocaine, cocaine, cyclomethycaine, dimethocaine,editocaine, hexylcaine, isobucaine, levobupivacaine, lidocaine,metabutethamine, metabutoxycaine, mepivacaine, meprylcaine,propoxycaine, prilocaine, procaine, piperocaine, tetracaine, trimecaine,and the like. The local injectable anesthetic compounds can havemultiple benefits in protein therapeutic formulations, such as reducedviscosity, improved stability, and reduced pain upon injection. In someembodiments, the local anesthetic compound is present in the formulationin a concentration of about 50 mg/ml or less.

In embodiments, a hindered amine having independent pharmacologicalproperties is used as an excipient compound in accordance with theformulations and methods described herein. In some embodiments, theexcipient compounds possessing independent pharmacological propertiesare present in an amount that does not have a pharmacological effectand/or that is not therapeutically effective. In other embodiments, theexcipient compounds possessing independent pharmacological propertiesare present in an amount that does have a pharmacological effect and/orthat is therapeutically effective. In certain embodiments, a hinderedamine having independent pharmacological properties is used incombination with another excipient compound that has been selected todecrease formulation viscosity, where the hindered amine havingindependent pharmacological properties is used to impart the benefits ofits pharmacological activity. For example, a local injectable anestheticcompound can be used to decrease formulation viscosity and also toreduce pain upon injection of the formulation. The reduction ofinjection pain can be caused by anesthetic properties; also a lowerinjection force can be required when the viscosity is reduced by theexcipients. Alternatively, a local injectable anesthetic compound can beused to impart the desirable pharmacological benefit of decreased localsensation during formulation injection, while being combined withanother excipient compound that reduces the viscosity of theformulation.

b. Excipient Compound Category 2: Anionic Aromatics

High concentration solutions of therapeutic or non-therapeutic proteinscan be formulated with anionic aromatic small molecule compounds asexcipient compounds. The anionic aromatic excipient compounds cancontain an aromatic functional group such as phenyl, benzyl, aryl,alkylbenzyl, hydroxybenzyl, phenolic, hydroxyaryl, heteroaromatic group,or a fused aromatic group. The anionic aromatic excipient compounds alsocan contain an anionic functional group such as carboxylate, oxide,phenoxide, sulfonate, sulfate, phosphonate, phosphate, or sulfide. Whilethe anionic aromatic excipients might be described as an acid, a sodiumsalt, or other, it is understood that the excipient can be used in avariety of salt forms. Without being bound by theory, an anionicaromatic excipient compound is thought to be a bulky, stericallyhindered molecule that can associate with cationic segments of aprotein, so that they can shield these sections of the protein, therebydecreasing the interactions between protein molecules that render theprotein-containing formulation viscous.

In embodiments, examples of anionic aromatic excipient compounds includecompounds such as salicylic acid, aminosalicylic acid, hydroxybenzoicacid, aminobenzoic acid, para-aminobenzoic acid, benzenesulfonic acid,hydroxybenzenesulfonic acid, naphthalenesulfonic acid,naphthalenedisulfonic acid, hydroquinone sulfonic acid, sulfanilic acid,vanillic acid, vanillin, vanillin-taurine adduct, aminophenol,anthranilic acid, cinnamic acid, coumaric acid, adenosine monophosphate,indole acetic acid, potassium urate, furan dicarboxylic acid,furan-2-acrylic acid, 2-furanpropionic acid, sodium phenylpyruvate,sodium hydroxyphenylpyruvate, dihydroxybenzoic acid, trihydroxybenzoicacid, pyrogallol, benzoic acid, and the salts of the foregoing acids. Inembodiments, the anionic aromatic excipient compounds is formulated inthe ionized salt form. In embodiments, an anionic aromatic compound isformulated as the salt of a hindered amine, such asdimethylcyclohexylammonium hydroxybenzoate. In embodiments, the anionicaromatic excipient compounds is formulated with various counterions suchas organic cations. In embodiments, high concentration solutions oftherapeutic or non-therapeutic proteins is formulated with anionicaromatic excipient compounds and caffeine. In embodiments, the anionicaromatic excipient compounds is metabolized in the body to yieldbiologically compatible byproducts.

c. Excipient Compound Category 3: Functionalized Amino Acids

High concentration solutions of therapeutic or non-therapeutic proteinscan be formulated with one or more functionalized amino acids, where asingle functionalized amino acid or an oligopeptide comprising one ormore functionalized amino acids may be used as the excipient compound.In embodiments, the functionalized amino acid compounds comprisemolecules (“amino acid precursors”) that can be hydrolyzed ormetabolized to yield amino acids. In embodiments, the functionalizedamino acids can contain an aromatic functional group such as phenyl,benzyl, aryl, alkylbenzyl, hydroxybenzyl, hydroxyaryl, heteroaromaticgroup, or a fused aromatic group. In embodiments, the functionalizedamino acid compounds can contain esterified amino acids, such as methyl,ethyl, propyl, butyl, benzyl, cycloalkyl, glyceryl, hydroxyethyl,hydroxypropyl, PEG, and PPG esters. In embodiments, the functionalizedamino acid compounds are selected from the group consisting of arginineethyl ester, arginine methyl ester, arginine hydroxyethyl ester, andarginine hydroxypropyl ester. In embodiments, the functionalized aminoacid compound is a charged ionic compound in aqueous solution at neutralpH. For example, a single amino acid can be derivatized by forming anester, like an acetate or a benzoate, and the hydrolysis products wouldbe acetic acid or benzoic acid, both natural materials, plus the aminoacid. In embodiments, the functionalized amino acid excipient compoundsis metabolized in the body to yield biologically compatible byproducts.

d. Excipient Compound Category 4: Oligopeptides

High concentration solutions of therapeutic or non-therapeutic proteinscan be formulated with oligopeptides as excipient compounds. Inembodiments, the oligopeptide is designed such that the structure has acharged section and a bulky section. In embodiments, the oligopeptidesconsist of between 2 and 10 peptide subunits. The oligopeptide can bebi-functional, for example a cationic amino acid coupled to a non-polarone, or an anionic one coupled to a non-polar one. In embodiments, theoligopeptides consist of between 2 and 5 peptide subunits. Inembodiments, the oligopeptides are homopeptides such as polyglutamicacid, polyaspartic acid, poly-lysine, poly-arginine, and poly-histidine.In embodiments, the oligopeptides have a net cationic charge. In otherembodiments, the oligopeptides are heteropeptides, such as Trp2Lys3. Inembodiments, the oligopeptide can have an alternating structure such asan ABA repeating pattern. In embodiments, the oligopeptide can containboth anionic and cationic amino acids, for example, Arg-Glu. Withoutbeing bound by theory, the oligopeptides comprise structures that canassociate with proteins in such a way that it reduces the intermolecularinteractions that lead to high viscosity solutions; for example, theoligopeptide-protein association can be a charge-charge interaction,leaving a somewhat non-polar amino acid to disrupt hydrogen bonding ofthe hydration layer around the protein, thus lowering viscosity. In someembodiments, the oligopeptide excipient is present in the composition ina concentration of about 50 mg/ml or less.

e. Excipient Compound Category 5: Short-Chain Organic Acids

As used herein, the term “short-chain organic acids” refers to C2-C6organic acid compounds and the salts, esters, or lactones thereof. Thiscategory includes saturated and unsaturated carboxylic acids, hydroxyfunctionalized carboxylic acids, and linear, branched, or cycliccarboxylic acids. In embodiments, the acid group in the short-chainorganic acid is a carboxylic acid, sulfonic acid, phosphonic acid, or asalt thereof.

In addition to the four excipient categories above, high concentrationsolutions of therapeutic or non-therapeutic proteins can be formulatedwith short-chain organic acids, for example, the acid or salt forms ofsorbic acid, valeric acid, propionic acid, caproic acid, and ascorbicacid as excipient compounds. Examples of excipient compounds in thiscategory include potassium sorbate, taurine, calcium propionate,magnesium propionate, and sodium ascorbate.

f. Excipient Compound Category 6: Low Molecular Weight AliphaticPolyacids

High concentration solutions of therapeutic or non-therapeutic PEGylatedproteins can be formulated with certain excipient compounds that enablelower solution viscosity, where such excipient compounds are lowmolecular weight aliphatic polyacids. As used herein, the term “lowmolecular weight aliphatic polyacids” refers to organic aliphaticpolyacids having a molecular weight <about 1500, and having at least twoacidic groups, where an acidic group is understood to be aproton-donating moiety. Non-limiting examples of acidic groups includecarboxylate, phosphonate, phosphate, sulfonate, sulfate, nitrate, andnitrite groups. Acidic groups on the low molecular weight aliphaticpolyacid can be in the anionic salt form such as carboxylate,phosphonate, phosphate, sulfonate, sulfate, nitrate, and nitrite; theircounterions can be sodium, potassium, lithium, and ammonium. Specificexamples of low molecular weight aliphatic polyacids useful forinteracting with PEGylated proteins as described herein include maleicacid, tartaric acid, glutaric acid, malonic acid, citric acid,ethylenediaminetetraacetic acid (EDTA), aspartic acid, glutamic acid,alendronic acid, etidronic acid and salts thereof. Further examples oflow molecular weight aliphatic polyacids in their anionic salt forminclude phosphate (PO₄ ³⁻), hydrogen phosphate (HPO₄ ³⁻), dihydrogenphosphate (H₂PO₄ ⁻), sulfate (SO₄ ²⁻), bisulfate (HSO₄ ⁻), pyrophosphate(P₂O₇ ⁴⁻), carbonate (CO₃ ²⁻), and bicarbonate (HCO₃ ⁻). The counterionfor the anionic salts can be Na, Li, K, or ammonium ion. Theseexcipients can also be used in combination with excipients. As usedherein, the low molecular weight aliphatic polyacid can also be an alphahydroxy acid, where there is a hydroxyl group adjacent to a first acidicgroup, for example glycolic acid, lactic acid, and gluconic acid andsalts thereof. In embodiments, the low molecular weight aliphaticpolyacid is an oligomeric form that bears more than two acidic groups,for example polyacrylic acid, polyphosphates, polypeptides and saltsthereof. In some embodiments, the low molecular weight aliphaticpolyacid excipient is present in the composition in a concentration ofabout 50 mg/ml or less.

5. Protein/Excipient Solutions: Properties and Processes

In certain embodiments, solutions of therapeutic or non-therapeuticproteins is formulated with the above-identified excipient compounds,such as hindered amines, anionic aromatics, functionalized amino acids,oligopeptides, short-chain organic acids to result in an improvedprotein-protein interaction characteristics as measured by the proteindiffusion interaction parameter, kD, or the second virial coefficient,B22. As used herein, an “improvement” in protein-protein interactioncharacteristics achieved by formulations using the above-identifiedexcipient compounds means a decrease in protein-protein interactions.These measurements of kD and B22 can be made using standard techniquesin the industry, and can be an indicator of improved solution propertiesor stability of the protein in solution. For example, a highly negativekD value can indicate that the protein has a strong attractiveinteraction and this can lead to aggregation, instability, and rheologyproblems. When formulated in the presence of certain of the aboveidentified excipient compounds, the same protein can have a lessnegative kD value, or a kD value near or above zero.

In embodiments, certain of the above-described excipient compounds, suchas hindered amines, anionic aromatics, functionalized amino acids,oligopeptides, short-chain organic acids, and/or low molecular weightaliphatic polyacids are used to improve a protein-related process, suchas the manufacture, processing, sterile filling, purification, andanalysis of protein-containing solutions, using processing methods suchas filtration, syringing, transferring, pumping, mixing, heating orcooling by heat transfer, gas transfer, centrifugation, chromatography,membrane separation, centrifugal concentration, tangential flowfiltration, radial flow filtration, axial flow filtration,lyophilization, and gel electrophoresis. These processes and processingmethods can have improved efficiency due to the lower viscosity,improved solubility, or improved stability of the proteins in thesolution during manufacture, processing, purification, and analysissteps. Additionally, equipment-related processes such as the cleanup,sterilization, and maintenance of protein processing equipment can befacilitated by the use of the above-identified excipients due todecreased fouling, decreased denaturing, lower viscosity, and improvedsolubility of the protein.

High concentration solutions of therapeutic proteins formulated with theabove described excipient compounds can be administered to patientsusing pre-filled syringes.

EXAMPLES

Materials:

-   -   Bovine gamma globulin (BGG), >99% purity, Sigma Aldrich    -   Histidine, Sigma Aldrich    -   Other materials described in the examples below were from Sigma        Aldrich unless otherwise specified.

Example 1: Preparation of Formulations Containing Excipient Compoundsand Test Protein

Formulations were prepared using an excipient compound and a testprotein, where the test protein was intended to simulate either atherapeutic protein that would be used in a therapeutic formulation, ora non-therapeutic protein that would be used in a non-therapeuticformulation. Such formulations were prepared in 50 mM histidinehydrochloride with different excipient compounds for viscositymeasurement in the following way. Histidine hydrochloride was firstprepared by dissolving 1.94 g histidine (Sigma-Aldrich, St. Louis, Mo.)in distilled water and adjusting the pH to about 6.0 with 1 Mhydrochloric acid (Sigma-Aldrich, St. Louis, Mo.) and then diluting to afinal volume of 250 mL with distilled water in a volumetric flask.Excipient compounds were then dissolved in 50 mM histidine HCl. Lists ofexcipients are provided below in Examples 4, 5, 6, and 7. In some casesexcipient compounds were adjusted to pH 6 prior to dissolving in 50 mMhistidine HCl. In this case the excipient compounds were first dissolvedin deionized water at about 5 wt % and the pH was adjusted to about 6.0with either hydrochloric acid or sodium hydroxide. The prepared saltsolution was then placed in a convection laboratory oven at about 150degrees Fahrenheit (about 65 degrees C.) to evaporate the water andisolate the solid excipient. Once excipient solutions in 50 mM histidineHCl had been prepared, the test protein (bovine gamma globulin (BGG)(Sigma-Aldrich, St. Louis, Mo.)) was dissolved at a ratio of about 0.336g BGG per 1 mL excipient solution. This resulted in a final proteinconcentration of about 280 mg/mL. Solutions of BGG in 50 mM histidineHCl with excipient were formulated in 20 mL vials and allowed to shakeat 100 rpm on an orbital shaker table overnight. BGG solutions were thentransferred to 2 mL microcentrifuge tubes and centrifuged for tenminutes at 2300 rpm in an IEC MicroMax microcentrifuge to removeentrained air prior to viscosity measurement.

Example 2: Viscosity Measurement

Viscosity measurements of formulations prepared as described in Example1 were made with a DV-IIT LV cone and plate viscometer (BrookfieldEngineering, Middleboro, Mass.). The viscometer was equipped with aCP-40 cone and was operated at 3 rpm and 25 degrees C. The formulationwas loaded into the viscometer at a volume of 0.5 mL and allowed toincubate at the given shear rate and temperature for 3 minutes, followedby a measurement collection period of twenty seconds. This was thenfollowed by 2 additional steps consisting of 1 minute of shearincubation and subsequent twenty-second measurement collection period.The three data points collected were then averaged and recorded as theviscosity for the sample.

Example 3: Protein Concentration Measurement

The concentration of the protein in the experimental solutions wasdetermined by measuring the absorbance of the protein solution at awavelength of 280 nm in a UV/VIS Spectrometer (Perkin Elmer Lambda 35).First the instrument was calibrated to zero absorbance with a 50 mMhistidine buffer at pH 6. Next the protein solutions were diluted by afactor of 300 with the same histidine buffer and the absorbance at 280nm recorded. The final concentration of the protein in the solution wascalculated by using the extinction coefficient value of 1.264mL/(mg×cm).

Example 4: Formulations with Hindered Amine Excipient Compounds

Formulations Containing 280 mg/mL BGG were Prepared as Described inExample 1, with some samples containing added excipient compounds. Inthese tests, the hydrochloride salts of dimethylcyclohexylamine (DMCHA),dicyclohexylmethylamine (DCHMA), dimethylaminopropylamine (DMAPA),triethanolamine (TEA), dimethylethanolamine (DMEA), and niacinamide weretested as examples of the hindered amine excipient compounds. Also ahydroxybenzoic acid salt of DMCHA and a taurine-dicyandiamide adductwere tested as examples of the hindered amine excipient compounds. Theviscosity of each protein solution was measured as described in Example2, and the results are presented in Table 1 below, showing the benefitof the added excipient compounds in reducing viscosity.

TABLE 1 Excipient Test Concentration Viscosity Viscosity NumberExcipient Added (mg/mL) (cP) Reduction 4.1 None 0 79 0% 4.2 DMCHA-HCl 2850 37% 4.3 DMCHA-HCl 41 43 46% 4.4 DMCHA-HCl 50 45 43% 4.5 DMCHA-HCl 8236 54% 4.6 DMCHA-HCl 123 35 56% 4.7 DMCHA-HCl 164 40 49% 4.8 DMAPA-HCl87 57 28% 4.9 DMAPA-HCl 40 54 32% 4.10 DCHMA-HCl 29 51 35% 4.11DCHMA-HCl 50 51 35% 4.14 TEA-HCl 97 51 35% 4.15 TEA-HCl 38 57 28% 4.16DMEA-HCl 51 51 35% 4.17 DMEA-HCl 98 47 41% 4.20 DMCHA- 67 46 42%hydroxybenzoate 4.21 DMCHA- 92 42 47% hydroxybenzoate 4.22 Product ofExample 8 26 58 27% 4.23 Product of Example 8 58 50 37% 4.24 Product ofExample 8 76 49 38% 4.25 Product of Example 8 103 46 42% 4.26 Product ofExample 8 129 47 41% 4.27 Product of Example 8 159 42 47% 4.28 Productof Example 8 163 42 47% 4.29 Niacinamide 48 39 51% 4.30 N-Methyl-2- 3045 43% pyrrolidone 4.31 N-Methyl-2- 52 52 34% pyrrolidone

Example 5: Formulations with Anionic Aromatic Excipient Compounds

Formulations of 280 mg/mL BGG were prepared as described in Example 1,with some samples containing added excipient compounds. The viscosity ofeach solution was measured as described in Example 2, and the resultsare presented in Table 2 below, showing the benefit of the addedexcipient compounds in reducing viscosity.

TABLE 2 Excipient Test Concentration Viscosity Viscosity NumberExcipient Added (mg/mL) (cP) Reduction 5.1 None 0 79 0% 5.2 Sodium 43 4839% aminobenzoate 5.3 Sodium 26 50 37% hydroxybenzoate 5.4 Sodiumsulfanilate 44 49 38% 5.5 Sodium sulfanilate 96 42 47% 5.6 Sodium indoleacetate 52 58 27% 5.7 Sodium indole acetate 27 78 1% 5.8 Vanillic acid,25 56 29% sodium salt 5.9 Vanillic acid, 50 50 37% sodium salt 5.10Sodium salicylate 25 57 28% 5.11 Sodium salicylate 50 52 34% 5.12Adenosine 26 47 41% monophosphate 5.13 Adenosine 50 66 16% monophosphate5.14 Sodium benzoate 31 61 23% 5.15 Sodium benzoate 56 62 22%

Example 6: Formulations with Oligopeptide Excipient Compounds

Oligopeptides (n=5) were synthesized by NeoBioLab Inc. in >95% puritywith the N terminus as a free amine and the C terminus as a free acid.Dipeptides (n=2) were synthesized by LifeTein LLC in 95% purity.Formulations of 280 mg/mL BGG were prepared as described in Example 1,with some samples containing the synthetic oligopeptides as addedexcipient compounds. The viscosity of each solution was measured asdescribed in Example 2, and the results are presented in Table 3 below,showing the benefit of the added excipient compounds in reducingviscosity.

TABLE 3 Excipient Con- Test centration Viscosity Viscosity NumberExcipient Added (mg/mL) (cP) Reduction 6.1 None 0 79 0% 6.2 ArgX5 100 5530% 6.3 ArgX5 50 54 32% 6.4 HisX5 100 62 22% 6.5 HisX5 50 51 35% 6.6HisX5 25 60 24% 6.7 Trp2Lys3 100 59 25% 6.8 Trp2Lys3 50 60 24% 6.9 AspX5100 102 −29% 6.10 AspX5 50 82 −4% 6.11 Dipeptide LE (Leu-Glu) 50 72 9%6.12 Dipeptide YE (Tyr-Glu) 50 55 30% 6.13 Dipeptide RP (Arg-Pro) 50 5135% 6.14 Dipeptide RK (Arg-Lys) 50 53 33% 6.15 Dipeptide RH (Arg-His) 5052 34% 6.16 Dipeptide RR (Arg-Arg) 50 57 28% 6.17 Dipeptide RE (Arg-Glu)50 50 37% 6.18 Dipeptide LE (Leu-Glu) 100 87 −10% 6.19 Dipeptide YE(Tyr-Glu) 100 68 14% 6.20 Dipeptide RP (Arg-Pro) 100 53 33% 6.21Dipeptide RK (Arg-Lys) 100 64 19% 6.22 Dipeptide RH (Arg-His) 100 72 9%6.23 Dipeptide RR (Arg-Arg) 100 62 22% 6.24 Dipeptide RE (Arg-Glu) 10066 16%

Example 8: Synthesis of Guanyl Taurine Excipient

Guanyl taurine was prepared following method described in U.S. Pat. No.2,230,965. Taurine (Sigma-Aldrich, St. Louis, Mo.) 3.53 parts were mixedwith 1.42 parts of dicyandiamide (Sigma-Aldrich, St. Louis, Mo.) andgrinded in a mortar and pestle until a homogeneous mixture was obtained.Next the mixture was placed in a flask and heated at 200° C. for 4hours. The product was used without further purification.

Example 9: Protein Formulations Containing Excipient Compounds

Formulations were prepared using an excipient compound and a testprotein, where the test protein was intended to simulate either atherapeutic protein that would be used in a therapeutic formulation, ora non-therapeutic protein that would be used in a non-therapeuticformulation. Such formulations were prepared in 50 mM aqueous histidinehydrochloride buffer solution with different excipient compounds forviscosity measurement in the following way. Histidine hydrochloridebuffer solution was first prepared by dissolving 1.94 g histidine(Sigma-Aldrich, St. Louis, Mo.) in distilled water and adjusting the pHto about 6.0 with 1 M hydrochloric acid (Sigma-Aldrich, St. Louis, Mo.)and then diluting to a final volume of 250 mL with distilled water in avolumetric flask. Excipient compounds were then dissolved in the 50 mMhistidine HCl buffer solution. A list of the excipient compounds isprovided in Table 4. In some cases excipient compounds were dissolved in50 mM histidine HCl and the resulting solution pH was adjusted withsmall amounts of concentrated sodium hydroxide or hydrochloric acid toachieve pH 6 prior to dissolution of the model protein. In some casesexcipient compounds were adjusted to pH 6 prior to dissolving in 50 mMhistidine HCl. In this case the excipient compounds were first dissolvedin deionized water at about 5 wt % and the pH was adjusted to about 6.0with either hydrochloric acid or sodium hydroxide. The prepared saltsolution was then placed in a convection laboratory oven at about 150degrees Fahrenheit (65 degrees C.) to evaporate the water and isolatethe solid excipient. Once excipient solutions in 50 mM histidine HCl hadbeen prepared, the test protein, bovine gamma globulin (Sigma-Aldrich,St. Louis, Mo.) was dissolved at a ratio to achieve a final proteinconcentration of about 280 mg/mL. Solutions of BGG in 50 mM histidineHCl with excipient were formulated in 20 mL vials and allowed to shakeat 100 rpm on an orbital shaker table overnight. BGG solutions were thentransferred to 2 mL microcentrifuge tubes and centrifuged for tenminutes at 2300 rpm in an IEC MicroMax microcentrifuge to removeentrained air prior to viscosity measurement.

Viscosity measurements of formulations prepared as described above weremade with a DV-IIT LV cone and plate viscometer (Brookfield Engineering,Middleboro, Mass.). The viscometer was equipped with a CP-40 cone andwas operated at 3 rpm and 25 degrees C. The formulation was loaded intothe viscometer at a volume of 0.5 mL and allowed to incubate at thegiven shear rate and temperature for 3 minutes, followed by ameasurement collection period of twenty seconds. This was then followedby 2 additional steps consisting of 1 minute of shear incubation andsubsequent twenty-second measurement collection period. The three datapoints collected were then averaged and recorded as the viscosity forthe sample. Viscosities of solutions with excipient were normalized tothe viscosity of the model protein solution without excipient. Thenormalized viscosity is the ratio of the viscosity of the model proteinsolution with excipient to the viscosity of the model protein solutionwith no excipient.

TABLE 4 Excipient Normalized Concentration Viscosity Viscosity TestNumber Excipient Added (mg/mL) (cP) Reduction 9.1 DMCHA-HCl 120 0.44 56%9.2 Niacinamide 50 0.51 49% 9.3 Isonicotinamide 50 0.48 52% 9.4 TyramineHCl 70 0.41 59% 9.5 Histamine HCl 50 0.41 59% 9.6 Imidazole HCl 100 0.4357% 9.7 2-methyl-2-imidazoline HCl 60 0.43 57% 9.81-butyl-3-methylimidazolium 100 0.48 52% chloride 9.9 Procaine HCl 500.53 47% 9.10 3-aminopyridine 50 0.51 49% 9.11 2,4,6-trimethylpyridine50 0.49 51% 9.12 3-pyridine methanol 50 0.53 47% 9.13 Nicotinamideadenine 20 0.56 44% dinucleotide 9.15 Sodium phenylpyruvate 55 0.57 43%9.16 2-Pyrrolidinone 60 0.68 32% 9.17 Morpholine HCl 50 0.60 40% 9.18Agmatine sulfate 55 0.77 23% 9.19 1-butyl-3-methylimidazolium 60 0.6634% iodide 9.21 L-Anserine nitrate 50 0.79 21% 9.221-hexyl-3-methylimidazolium 65 0.89 11% chloride 9.23 N,N-diethylnicotinamide 50 0.67 33% 9.24 Nicotinic acid, sodium salt 100 0.54 46%9.25 Biotin 20 0.69 31%

Example 10: Preparation of Formulations Containing ExcipientCombinations and Test Protein

Formulations were prepared using a primary excipient compound, asecondary excipient compound and a test protein, where the test proteinwas intended to simulate either a therapeutic protein that would be usedin a therapeutic formulation, or a non-therapeutic protein that would beused in a non-therapeutic formulation. The primary excipient compoundswere selected from compounds having both anionic and aromaticfunctionality, as listed below in Table 5. The secondary excipientcompounds were selected from compounds having either nonionic orcationic charge at pH 6 and either imidazoline or benzene rings, aslisted below in Table 5. Formulations of these excipients were preparedin 50 mM histidine hydrochloride buffer solution for viscositymeasurement in the following way. Histidine hydrochloride was firstprepared by dissolving 1.94 g histidine (Sigma-Aldrich, St. Louis, Mo.)in distilled water and adjusting the pH to about 6.0 with 1 Mhydrochloric acid (Sigma-Aldrich, St. Louis, Mo.) and then diluting to afinal volume of 250 mL with distilled water in a volumetric flask. Theindividual primary or secondary excipient compounds were then dissolvedin 50 mM histidine HCl. Combinations of primary and secondary excipientswere dissolved in 50 mM histidine HCl and the resulting solution pHadjusted with small amounts of concentrated sodium hydroxide orhydrochloric acid to achieve pH 6 prior to dissolution of the modelprotein. Once excipient solutions had been prepared as described above,the test protein (bovine gamma globulin (BGG) (Sigma-Aldrich, St. Louis,Mo.) was dissolved into each test solution at a ratio to achieve a finalprotein concentration of about 280 mg/mL. Solutions of BGG in 50 mMhistidine HCl with excipient were formulated in 20 mL vials and allowedto shake at 100 rpm on an orbital shaker table overnight. BGG solutionswere then transferred to 2 mL microcentrifuge tubes and centrifuged forten minutes at 2300 rpm in an IEC MicroMax microcentrifuge to removeentrained air prior to viscosity measurement.

Viscosity measurements of formulations prepared as described above weremade with a DV-IIT LV cone and plate viscometer (Brookfield Engineering,Middleboro, Mass.). The viscometer was equipped with a CP-40 cone andwas operated at 3 rpm and 25 degrees C. The formulation was loaded intothe viscometer at a volume of 0.5 mL and allowed to incubate at thegiven shear rate and temperature for 3 minutes, followed by ameasurement collection period of twenty seconds. This was then followedby 2 additional steps consisting of 1 minute of shear incubation and asubsequent twenty-second measurement collection period. The three datapoints collected were then averaged and recorded as the viscosity forthe sample. Viscosities of solutions with excipient were normalized tothe viscosity of the model protein solution without excipient, andsummarized in Table 5 below. The normalized viscosity is the ratio ofthe viscosity of the model protein solution with excipient to theviscosity of the model protein solution with no excipient. The exampleshows that a combination of primary and secondary excipients can give abetter result than a single excipient.

TABLE 5 Primary Excipient Secondary Excipient Test ConcentrationConcentration Normalized Number Name (mg/mL) Name (mg/mL) Viscosity 10.1Salicylic Acid 30 None 0 0.79 10.2 Salicylic Acid 25 Imidazole 4 0.5910.3 4-hydroxybenzoic 30 None 0 0.61 acid 10.4 4-hydroxybenzoic 25Imidazole 5 0.57 acid 10.5 4-hydroxybenzene 31 None 0 0.59 sulfonic acid10.6 4-hydroxybenzene 26 Imidazole 5 0.70 sulfonic acid 10.74-hydroxybenzene 25 Caffeine 5 0.69 sulfonic acid 10.8 None 0 Caffeine10 0.73 10.9 None 0 Imidazole 5 0.75

Example 11: Preparation of Formulations Containing ExcipientCombinations and Test Protein

Formulations were prepared using a primary excipient compound, asecondary excipient compound and a test protein, where the test proteinwas intended to simulate a therapeutic protein that would be used in atherapeutic formulation, or a non-therapeutic protein that would be usedin a non-therapeutic formulation. The primary excipient compounds wereselected from compounds having both anionic and aromatic functionality,as listed below in Table 6. The secondary excipient compounds wereselected from compounds having either nonionic or cationic charge at pH6 and either imidazoline or benzene rings, as listed below in Table 6.Formulations of these excipients were prepared in distilled water forviscosity measurement in the following way. Combinations of primary andsecondary excipients were dissolved in distilled water and the resultingsolution pH adjusted with small amounts of concentrated sodium hydroxideor hydrochloric acid to achieve pH 6 prior to dissolution of the modelprotein. Once excipient solutions in distilled water had been prepared,the test protein (bovine gamma globulin (BGG) (Sigma-Aldrich, St. Louis,Mo.)) was dissolved at a ratio to achieve a final protein concentrationof about 280 mg/mL. Solutions of BGG in distilled water with excipientwere formulated in 20 mL vials and allowed to shake at 100 rpm on anorbital shaker table overnight. BGG solutions were then transferred to 2mL microcentrifuge tubes and centrifuged for ten minutes at 2300 rpm inan IEC MicroMax microcentrifuge to remove entrained air prior toviscosity measurement.

Viscosity measurements of formulations prepared as described above weremade with a DV-IIT LV cone and plate viscometer (Brookfield Engineering,Middleboro, Mass.). The viscometer was equipped with a CP-40 cone andwas operated at 3 rpm and 25 degrees C. The formulation was loaded intothe viscometer at a volume of 0.5 mL and allowed to incubate at thegiven shear rate and temperature for 3 minutes, followed by ameasurement collection period of twenty seconds. This was then followedby 2 additional steps consisting of 1 minute of shear incubation and asubsequent twenty-second measurement collection period. The three datapoints collected were then averaged and recorded as the viscosity forthe sample. Viscosities of solutions with excipient were normalized tothe viscosity of the model protein solution without excipient, andsummarized in Table 6 below. The normalized viscosity is the ratio ofthe viscosity of the model protein solution with excipient to theviscosity of the model protein solution with no excipient. The exampleshows that a combination of primary and secondary excipients can give abetter result than a single excipient.

TABLE 6 Primary Excipient Secondary Excipient Test ConcentrationConcentration Normalized Number Name (mg/mL) Name (mg/mL) Viscosity 11.1Salicylic Acid 20 None 0 0.96 11.2 Salicylic Acid 20 Caffeine 5 0.7111.3 Salicylic Acid 20 Niacinamide 5 0.76 11.4 Salicylic Acid 20Imidazole 5 0.73

Example 12: Preparation of Formulations Containing Excipient Compoundsand PEG

Materials: All materials were purchased from Sigma-Aldrich, St. Louis,Mo. Formulations were prepared using an excipient compound and PEG,where the PEG was intended to simulate a therapeutic PEGylated proteinthat would be used in a therapeutic formulation. Such formulations wereprepared by mixing equal volumes of a solution of PEG with a solution ofthe excipient. Both solutions were prepared in a Tris buffer consistingof 10 mM Tris, 135 mM NaCl, 1 mM trans-cinnamic acid at pH of 7.3.

The PEG solution was prepared by mixing 3 g of Poly(ethylene oxide)average Mw˜1,000,000 (Aldrich Catalog #372781) with 97 g of the Trisbuffer solution. The mixture was stirred overnight for completedissolution.

An example of the excipient solution preparation is as follows: Anapproximately 80 mg/mL solution of citric acid in the Tris buffer wasprepared by dissolving 0.4 g of citric acid (Aldrich cat. #251275) in 5mL of the Tris buffer solution and adjusted the pH to 7.3 with minimumamount of 10 M NaOH solution.

The PEG excipient solution was prepared by mixing 0.5 mL of the PEGsolution with 0.5 mL of the excipient solution and mixed by using avortex for a few seconds. A control sample was prepared by mixing 0.5 mLof the PEG solution with 0.5 mL of the Tris buffer solution.

Example 13: Viscosity Measurements of Formulations Containing ExcipientCompounds and PEG

Viscosity measurements of the formulations prepared were made with aDV-IIT LV cone and plate viscometer (Brookfield Engineering, Middleboro,Mass.). The viscometer was equipped with a CP-40 cone and was operatedat 3 rpm and 25 degrees C. The formulation was loaded into theviscometer at a volume of 0.5 mL and allowed to incubate at the givenshear rate and temperature for 3 minutes, followed by a measurementcollection period of twenty seconds. This was then followed by 2additional steps consisting of 1 minute of shear incubation andsubsequent twenty second measurement collection period. The three datapoints collected were then averaged and recorded as the viscosity forthe sample.

The results presented in Table 7 show the effect of the added excipientcompounds in reducing viscosity.

TABLE 7 Excipient Test Concentration Viscosity Viscosity NumberExcipient (mg/mL) (cP) Reduction 13.1 None 0 104.8 0% 13.2 Citric acidNa salt 40 56.8 44% 13.3 Citric acid Na salt 20 73.3 28% 13.4 glycerolphosphate 40 71.7 30% 13.5 glycerol phosphate 20 83.9 18% 13.6 Ethylenediamine 40 84.7 17% 13.7 Ethylene diamine 20 83.9 15% 13.8 EDTA/K salt40 67.1 36% 13.9 EDTA/K salt 20 76.9 27% 13.10 EDTA/Na salt 40 68.1 35%13.11 EDTA/Na salt 20 77.4 26% 13.12 D-Gluconic acid/K salt 40 80.32 23%13.13 D-Gluconic acid/K salt 20 88.4 16% 13.14 D-Gluconic acid/ 40 81.2423% Na salt 13.15 D-Gluconic acid/ 20 86.6 17% Na salt 13.16 lacticacid/K salt 40 80.42 23% 13.17 lactic acid/K salt 85.1 19% 13.18 lacticacid/Na salt 40 86.55 17% 13.19 lactic acid/Na salt 20 87.2 17% 13.20etidronic acid/K salt 24 71.91 31% 13.21 etidronic acid/K salt 12 80.523% 13.22 etidronic acid/Na salt 24 71.6 32% 13.23 etidronic acid/Nasalt 12 79.4 24%

Example 14: Preparation of PEGylated BSA with 1 PEG Chain Per BSAMolecule

To a beaker was added 200 mL of a phosphate buffered saline (AldrichCat. #P4417) and 4 g of BSA (Aldrich Cat. #A7906) and mixed with amagnetic bar. Next 400 mg of methoxy polyethylene glycol maleimide,MW=5,000, (Aldrich Cat. #63187) was added. The reaction mixture wasallowed to react overnight at room temperature. The following day, 20drops of HCl 0.1 M were added to stop the reaction. The reaction productwas characterized by SDS-Page and SEC which clearly showed the PEGylatedBSA. The reaction mixture was placed in an Amicon centrifuge tube with amolecular weight cutoff (MWCO) of 30,000 and concentrated to a fewmilliliters. Next the sample was diluted 20 times with a histidinebuffer, 50 mM at a pH of approximately 6, followed by concentratinguntil a high viscosity fluid was obtained. The final concentration ofthe protein solution was obtained by measuring the absorbance at 280 nmand using a coefficient of extinction for the BSA of 0.6678. The resultsindicated that the final concentration of BSA in the solution was 342mg/mL.

Example 15: Preparation of PEGylated BSA with Multiple PEG Chains PerBSA Molecule

A 5 mg/mL solution of BSA (Aldrich A7906) in phosphate buffer, 25 mM atpH of 7.2, was prepared by mixing 0.5 g of the BSA with 100 mL of thebuffer. Next 1 g of a methoxy PEG propionaldehyde Mw=20,000 (JenKemTechnology, Plano, Tex. 75024) was added followed by 0.12 g of sodiumcyanoborohydride (Aldrich 156159). The reaction was allowed to proceedovernight at room temperature. The following day the reaction mixturewas diluted 13 times with a Tris buffer (10 mM Tris, 135 mM NaCl atpH=7.3) and concentrated using Amicon centrifuge tubes MWCO of 30,000until a concentration of approximately 150 mg/mL was reached.

Example 16: Preparation of PEGylated Lysozyme with Multiple PEG ChainsPer Lysozyme Molecule

A 5 mg/mL solution of lysozyme (Aldrich L6876) in phosphate buffer, 25mM at pH of 7.2, was prepared by mixing 0.5 g of the lysozyme with 100mL of the buffer. Next 1 g of a methoxy PEG propionaldehyde Mw=5,000(JenKem Technology, Plano, Tex. 75024) was added followed by 0.12 g ofSodium cyanoborohydride (Aldrich 156159). The reaction was allowed toproceed overnight at room temperature. The following day the reactionmixture was diluted 49 times with the phosphate buffer, 25 mM at pH of7.2, and concentrated using Amicon centrifuge tubes MWCO of 30,000. Thefinal concentration of the protein solution was obtained by measuringthe absorbance at 280 nm and using a coefficient of extinction for thelysozyme of 2.63. The final concentration of lysozyme in the solutionwas 140 mg/mL.

Example 17: Effect of Excipients on Viscosity of PEGylated BSA with 1PEG Chain Per BSA Molecule

Formulations of PEGylated BSA (from Example 14 above) with excipientswere prepared by adding 6 or 12 milligrams of the excipient salt to 0.3mL of the PEGylated BSA solution. The solution was mixed by gentlyshaking and the viscosity was measured by a RheoSense microVisc equippedwith an A10 channel (100 micron depth) at a shear rate of 500 sec-1. Theviscometer measurements were completed at ambient temperature.

The results presented in Table 8 shows the effect of the added excipientcompounds in reducing viscosity.

TABLE 8 Excipient Test Concentration Viscosity Viscosity NumberExcipient (mg/mL) (cP) Reduction 17.1 None 0 228.6 0% 17.2Alpha-Cyclodextrin 20 151.5 34% sulfated Na salt 17.3 K acetate 40 89.560%

Example 18: Effect of Excipients on Viscosity of PEGylated BSA withMultiple PEG Chains Per BSA Molecule

A formulation of PEGylated BSA (from Example 15 above) with citric acidNa salt as excipient was prepared by adding 8 milligrams of theexcipient salt to 0.2 mL of the PEGylated BSA solution. The solution wasmixed by gently shaking and the viscosity was measured by a RheoSensemicroVisc equipped with an A10 channel (100 micron depth) at a shearrate of 500 sec-1. The viscometer measurements were completed at ambienttemperature. The results presented in Table 9 shows the effect of theadded excipient compounds in reducing viscosity.

TABLE 9 Excipient Test Concentration Viscosity Viscosity NumberExcipient Added (mg/mL) (cP) Reduction 18.1 None 0 56.8 0% 18.2 Citricacid Na salt 40 43.5 23%

Example 19: Effect of Excipients on Viscosity of PEGylated Lysozyme withMultiple PEG Chains Per Lysozyme Molecule

A formulation of PEGylated lysozyme (from Example 16 above) withpotassium acetate as excipient was prepared by adding 6 milligrams ofthe excipient salt to 0.3 mL of the PEGylated lysozyme solution. Thesolution was mixed by gently shaking and the viscosity was measured by aRheoSense microVisc equipped with an A10 channel (100 micron depth) at ashear rate of 500 sec-1. The viscometer measurements were completed atambient temperature. The results presented in the next table shows thebenefit of the added excipient compounds in reducing viscosity.

TABLE 10 Excipient Concentration Viscosity Viscosity Test NumberExcipient (mg/mL) (cP) Reduction 19.1 None 0 24.6 0% 19.2 K acetate 2022.6 8%

Example 20: Protein Formulations Containing Excipient Combinations

Formulations were prepared using an excipient compound or a combinationof two excipient compounds and a test protein, where the test proteinwas intended to simulate a therapeutic protein that would be used in atherapeutic formulation. These formulations were prepared in 20 mMhistidine buffer with different excipient compounds for viscositymeasurement in the following way. Excipient combinations were dissolvedin 20 mM histidine (Sigma-Aldrich, St. Louis, Mo.) and the resultingsolution pH adjusted with small amounts of concentrated sodium hydroxideor hydrochloric acid to achieve pH 6 prior to dissolution of the modelprotein. Excipient compounds for this Example are listed below in Table11. Once excipient solutions had been prepared, the test protein (bovinegamma globulin or “BGG” (Sigma-Aldrich, St. Louis, Mo.)) was dissolvedat a ratio to achieve a final protein concentration of about 280 mg/mL.Solutions of BGG in the excipient solutions were formulated in 5 mLsterile polypropylene tubes and allowed to shake at 80-100 rpm on anorbital shaker table overnight. BGG solutions were then transferred to 2mL microcentrifuge tubes and centrifuged for about ten minutes at 2300rpm in an IEC MicroMax microcentrifuge to remove entrained air prior toviscosity measurement.

Viscosity measurements of formulations prepared as described above weremade with a DV-IIT LV cone and plate viscometer (Brookfield Engineering,Middleboro, Mass.). The viscometer was equipped with a CP-40 cone andwas operated at 3 rpm and 25 degrees Centigrade. The formulation wasloaded into the viscometer at a volume of 0.5 mL and allowed to incubateat the given shear rate and temperature for 3 minutes, followed by ameasurement collection period of twenty seconds. This was then followedby 2 additional steps consisting of 1 minute of shear incubation andsubsequent twenty second measurement collection period. The three datapoints collected were then averaged and recorded as the viscosity forthe sample. Viscosities of solutions with excipient were normalized tothe viscosity of the model protein solution without excipient, and theresults are shown in Table 11 below. The normalized viscosity is theratio of the viscosity of the model protein solution with excipient tothe viscosity of the model protein solution with no excipient.

TABLE 11 Excipient A Excipient B Test Conc. Conc. Normalized # Name(mg/mL) Name (mg/mL) Viscosity 20.1 None 0 None 0 1.00 20.2 Aspartame 10None 0 0.83 Saccharin 60 None 0 0.51 20.4 Acesulfame K 80 None 0 0.4420.5 Theophylline 10 None 0 0.84 20.6 Saccharin 30 None 0 0.58 20.7Acesulfame K 40 None 0 0.61 20.8 Caffeine 15 Taurine 15 0.82 20.9Caffeine 15 Tyramine 15 0.67

Example 21: Protein Formulations Containing Excipients to ReduceViscosity and Injection Pain

Formulations were prepared using an excipient compound, a secondexcipient compound, and a test protein, where the test protein wasintended to simulate a therapeutic protein that would be used in atherapeutic formulation. The first excipient compound, Excipient A, wasselected from a group of compounds having local anesthetic properties.The first excipient, Excipient A and the second excipient, Excipient Bare listed in Table 12. These formulations were prepared in 20 mMhistidine buffer using Excipient A and Excipient B in the following way,so that their viscosities could be measured. Excipients in the amountsdisclosed in Table 12 were dissolved in 20 mM histidine (Sigma-Aldrich,St Louis, Mo.) and the resulting solutions were pH adjusted with smallamounts of concentrated sodium hydroxide or hydrochloric acid to achievepH 6 prior to dissolution of the model protein. Once excipient solutionshad been prepared, the test protein (bovine gamma globulin (“BGG”)(Sigma-Aldrich, St. Louis, Mo.)) was dissolved in the excipient solutionat a ratio to achieve a final protein concentration of about 280 mg/mL.Solutions of BGG in the excipient solutions were formulated in 5 mLsterile polypropylene tubes and allowed to shake at 80-100 rpm on anorbital shaker table overnight. BGG-excipient solutions were thentransferred to 2 mL microcentrifuge tubes and centrifuged for about tenminutes at 2300 rpm in an IEC MicroMax microcentrifuge to removeentrained air prior to viscosity measurement.

Viscosity measurements of the formulations prepared as described abovewere made with a DV-IIT LV cone and plate viscometer (BrookfieldEngineering, Middleboro, Mass.). The viscometer was equipped with aCP-40 cone and was operated at 3 rpm and 25 degrees Centigrade. Theformulation was loaded into the viscometer at a volume of 0.5 mL andallowed to incubate at the given shear rate and temperature for 3minutes, followed by a measurement collection period of twenty seconds.This was then followed by 2 additional steps consisting of 1 minute ofshear incubation and subsequent twenty second measurement collectionperiod. The three data points collected were then averaged and recordedas the viscosity for the sample. Viscosities of solutions with excipientwere normalized to the viscosity of the model protein solution withoutexcipient, and the results are shown in Table 12 below. The normalizedviscosity is the ratio of the viscosity of the model protein solutionwith excipient to the viscosity of the model protein solution with noexcipient.

TABLE 12 Excipient A Excipient B Test Conc. Conc. Normalized # Name(mg/mL) Name (mg/mL) Viscosity 21.1 None 0 None 0 1.00 21.2 Lidocaine 45None 0 0.73 21.3 Lidocaine 23 None 0 0.74 21.4 Lidocaine 10 Caffeine 150.71 21.5 Procaine HCl 40 None 0 0.64 21.6 Procaine HCl 20 Caffeine 150.69

Example 22: Formulations Containing Excipient Compounds and PEG

Formulations were prepared using an excipient compound and PEG, wherethe PEG was intended to simulate a therapeutic PEGylated protein thatwould be used in a therapeutic formulation, and where the excipientcompounds were provided in the amounts as listed in Table 13. Theseformulations were prepared by mixing equal volumes of a solution of PEGwith a solution of the excipient. Both solutions were prepared inDI-Water.

The PEG solution was prepared by mixing 16.5 g of poly(ethylene oxide)average Mw˜100,000 (Aldrich Catalog #181986) with 83.5 g of DI water.The mixture was stirred overnight for complete dissolution.

The excipient solutions were prepared by this general method and asdetailed in Table 13 below: An approximately 20 mg/mL solution ofpotassium phosphate tribasic (Aldrich Catalog #P5629) in DI-water wasprepared by dissolving 0.05 g of potassium phosphate in 5 mL ofDI-water. The PEG excipient solution was prepared by mixing 0.5 mL ofthe PEG solution with 0.5 mL of the excipient solution and mixed byusing a vortex for a few seconds. A control sample was prepared bymixing 0.5 mL of the PEG solution with 0.5 mL of DI-water. Viscosity wasmeasured and results are recorded in Table 13 below.

TABLE 13 Excipient Viscosity Test Concentration Viscosity ReductionNumber Excipient (mg/mL) (cP) (%) 22.1 None  0 79.7 0 22.2 Citric acidNa salt 10 74.9 6.0 22.3 Potassium 10 72.3 9.3 phosphate 22.4 Citricacid Na 10/10 69.1 13.3 salt/Potassium phosphate 22.5 Sodium sulfate 1075.1 5.8 22.6 Citric acid Na 10/10 70.4 11.7 salt/Sodium sulfate

Example 23: Improved Processing of Protein Solutions with Excipients

Two BGG solutions were prepared by mixing 0.25 g of solid BGG (Aldrichcatalogue number G5009) with 4 ml of a buffer solution. For Sample A:Buffer solution was 20 mM histidine buffer (pH=6.0). For sample B:Buffer solution was 20 mM histidine buffer containing 15 mg/ml ofcaffeine (pH=6). The dissolution of the solid BGG was carried out byplacing the samples in an orbital shaker set at 100 rpm. The buffersample containing caffeine excipient was observed to dissolve theprotein faster. For the sample with the caffeine excipient (Sample B)complete dissolution of the BGG was achieved in 15 minutes. For thesample without the caffeine (Sample A) the dissolution needed 35minutes.

Next the samples were placed in 2 separate Amicon Ultra 4 CentrifugalFilter Unit with a 30,000 molecular weight cut off and the samples werecentrifuged at 2,500 rpm at 10 minutes intervals. The filtrate volumerecovered after each 10 minute centrifuge run was recorded. The resultsin Table 14 show the faster recovery of the filtrate for Sample B. Inaddition Sample B kept concentrating with every additional run butSample A reached a maximum concentration point and furthercentrifugation did not result in further sample concentration.

TABLE 14 Centrifuge Sample A filtrate collected Sample B filtratecollected time (min) (mL) (mL) 10 0.28 0.28 20 0.56 0.61 30 0.78 0.88 400.99 1.09 50 1.27 1.42 60 1.51 1.71 70 1.64 1.99 80 1.79 2.29 90 1.792.39 100 1.79 2.49

Example 24: Protein Formulations Containing Multiple Excipients

This example shows how the combination of caffeine and arginine asexcipients has a beneficial effect on decreasing viscosity of a BGGsolution. Four BGG solutions were prepared by mixing 0.18 g of solid BGG(Aldrich catalogue number G5009) with 0.5 mL of a 20 mM Histidine bufferat pH 6. Each buffer solution contained different excipient orcombination of excipients as described in the table below. The viscosityof the solutions was measured as described in previous examples. Theresults show that the hindered amine excipient, caffeine, can becombined with known excipients such as arginine, and the combination hasbetter viscosity reduction properties than the individual excipients bythemselves.

TABLE 15 Viscosity Viscosity Sample Excipient added (cP) Reduction (%) ANone 130.6 0 B Caffeine (10 mg/ml) 87.9 33 C Caffeine (10mg/ml)/Arginine 66.1 49 (25 mg/ml) D Arginine (25 mg/ml) 76.7 41

Arginine was added to 280 mg/mL solutions of BGG in histidine buffer atpH 6. At levels above 50 mg/mL, adding more arginine did not decreaseviscosity further, as shown in Table 16.

TABLE 16 Arginine added Viscosity Viscosity (mg/mL) (cP) reduction (%) 079.0 0% 53 40.9 48% 79 46.1 42% 105 47.8 40% 132 49.0 38% 158 48.0 39%174 50.3 36% 211 51.4 35%

Caffeine was added to 280 mg/mL solutions of BGG in histidine buffer atpH 6. At levels above 10 mg/ml, adding more caffeine did not decreaseviscosity further, as shown in Table 17.

TABLE 17 Caffeine added Viscosity Viscosity (mg/mL) (cP) reduction (%) 079 0% 10 60 31% 15 62 23% 22 50 45%

EQUIVALENTS

While specific embodiments of the subject invention have been disclosedherein, the above specification is illustrative and not restrictive.While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims. Many variations of the inventionwill become apparent to those of skilled art upon review of thisspecification. Unless otherwise indicated, all numbers expressingreaction conditions, quantities of ingredients, and so forth, as used inthis specification and the claims are to be understood as being modifiedin all instances by the term “about.” Accordingly, unless indicated tothe contrary, the numerical parameters set forth herein areapproximations that can vary depending upon the desired propertiessought to be obtained by the present invention.

What is claimed is:
 1. A method of reducing a viscosity of a liquidprotein formulation, comprising: preparing the liquid proteinformulation comprising a therapeutic protein and a viscosity-reducingamount of caffeine, wherein (i) the liquid protein formulation containsat least about 100 mg/mL of the therapeutic protein, and (ii) theviscosity-reducing amount of caffeine is about 30 mg/ml or less, whereinthe viscosity of the liquid protein formulation is at least about 50%less than a viscosity of a control formulation, and wherein the controlformulation does not contain caffeine but is otherwise identical on adry weight basis to the liquid protein formulation.
 2. The method ofclaim 1, wherein the viscosity-reducing amount of caffeine is about 22mg/ml or less.
 3. The method of claim 1, wherein the viscosity-reducingamount of caffeine is about 15 mg/ml or less.
 4. The method of claim 1,wherein the liquid protein formulation contains at least about 200 mg/mlof the therapeutic protein.
 5. The method of claim 1, wherein the liquidprotein formulation contains at least about 300 mg/ml of the therapeuticprotein.
 6. The method of claim 1, wherein the therapeutic protein is atherapeutic antibody.
 7. The method of claim 6, wherein the liquidprotein formulation contains at least about 200 mg/ml of the therapeuticantibody.
 8. The method of claim 7, wherein the liquid proteinformulation contains at least about 300 mg/ml of the therapeuticantibody.
 9. The method of claim 1, wherein the viscosity of the liquidprotein formulation is at least about 70% less than the viscosity of thecontrol formulation.
 10. The method of claim 1, wherein the viscosity ofthe liquid protein formulation is at least about 90% less than theviscosity of the control formulation.
 11. The method of claim 1, whereinthe viscosity of the liquid protein formulation is less than about 20cP.
 12. The method of claim 11, wherein the viscosity of the liquidprotein formulation is less than about 10 cP.
 13. The method of claim 1,wherein the liquid protein formulation comprises a secondviscosity-reducing excipient.
 14. The method of claim 13, wherein thesecond viscosity-reducing excipient is selected from the groupconsisting of theophylline, tyramine, procaine, lidocaine, imidazole,aspartame, saccharin, acesulfame potassium, niacinamide, andisonicotinamide.
 15. The method of claim 1, wherein the liquid proteinformulation is an injectable formulation.
 16. The method of claim 1,wherein the liquid protein formulation is an aqueous formulation. 17.The method of claim 1, wherein the viscosity-reducing amount of caffeineis about 5 mg/ml to about 15 mg/ml.